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Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres

  • Robert E. Hynds
  • Colin R. Butler
  • Sam M. Janes
  • Adam GiangrecoEmail author
Part of the Methods in Molecular Biology book series


Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy.


Lung Stem cells Epithelial cells Goblet cells Cilia Adult stem cells Cell culture techniques Primary cell culture 



We thank Prof. Richard Schlegel (Georgetown University, USA), Xuefeng Liu (Georgetown University, USA), and Dr. Henry Danahay (University of Sussex, UK) for sharing their laboratories protocols during the development of those described here.

This work was supported by a BBSRC-CASE studentship with industrial support from Unilever (R.E.H.), a Wellcome Trust Clinical Research Fellowship (C.R.B.), an ERC Starting Grant (A.G.), a Wellcome Trust Senior Fellowship (S.M.J.) and was undertaken at UCLH/UCL who receive funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme and the UCL Experimental Cancer Medicine Centre (S.M.J.).


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Robert E. Hynds
    • 1
  • Colin R. Butler
    • 1
  • Sam M. Janes
    • 1
  • Adam Giangreco
    • 1
    Email author
  1. 1.Lungs for Living Research Centre, UCL RespiratoryUniversity College LondonLondonUK

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